JP2019523652A5

JP2019523652A5 -

Info

Publication number: JP2019523652A5
Application number: JP2019513484A
Authority: JP
Filing date: 2017-05-17
Publication date: 2020-06-25
Anticipated expiration: 2037-05-17

Claims

A method of identifying the identities of multiple biological samples containing nucleic acids, comprising:
A step of preparing a plurality of carriers each containing a biological sample containing a nucleic acid, which is a substrate or a container,
Here, each carrier contains, in addition to the sample containing the nucleic acid, at least two nucleotide barcode nucleic acids for labeling the carrier,
Each of these at least two nucleotide barcode nucleic acids comprises a minimal nucleotide barcode sequence that differs by at least 4 nucleotides in length,
The combination of these different nucleotide barcode nucleic acids produces a movable molecular identification barcode such that each movable molecular identification barcode is different for each of the carriers,
Flanked on one or both sides of the minimal nucleotide barcode sequence in the at least two nucleotide barcode nucleic acids by non-viral and non-bacterial nucleotide barcode sequence identifier sequences that are identical in all nucleotide barcode nucleic acids,
Flanked on one or both sides by non-viral and non-bacterial extracted sequences that are identical in all nucleotide barcode nucleic acids, these allow the identification of said minimal nucleotide barcode sequences,
Each carrier comprises a macroscopic barcode label corresponding to a movable molecular identification barcode affixed to the carrier, and
Sequencing with one or more target sequences in the nucleic acid of the biological sample, the step of sequencing the target sequence in the nucleotide barcode nucleic acid comprising the minimal nucleotide barcode sequence,
Sequencing of the target sequence in the nucleic acid of the biological sample, and sequencing of the target sequence of the nucleotide barcode nucleic acid containing the minimal nucleotide barcode sequence is performed by a parallel sequencing method,
The parallel sequencing method, the target sequence of the nucleic acid of a biological sample, and proceeds by pooling target sequence of said nucleotide barcode nucleic acid comprising the minimum nucleotides barcode sequence, step, or in different samples Derived, proceeded by pooling the target sequence in the nucleic acid of the biological sample, and the target sequence of the nucleotide barcode nucleic acid comprising the minimal nucleotide barcode sequence,
From the obtained sequence data, the step of identifying and selecting the sequence derived from the nucleotide barcode nucleic acid, the step of selecting those sequences derived from the nucleotide barcode nucleic acid from the obtained sequence data Including,
The selecting step comprises identifying a sequence having one or more nucleotide barcode sequence identifier sequences adjacent to the minimal nucleotide barcode sequence, and
Identifying and selecting said minimal nucleotide barcode sequence within said isolated sequence having a nucleotide barcode sequence identifier sequence,
Selecting the sequence that is between the two extracted sequences at a defined length, or selects the sequence that is located adjacent to one extracted sequence at the defined length,
Identifying these selected sequences as minimal nucleotide barcode sequences,
By matching the identified minimal nucleotide barcode sequence with the expected minimal nucleotide barcode sequence based on the macroscopic barcode label provided by the carrier, the identity of the sample and/or Identifying the contamination,
Including the method.

With the step of connecting an adapter to the target nucleic acid in the sample, and to the nucleic acid containing the nucleotide barcode sequence,
The method of claim 1, comprising sequencing the target sequence and the nucleotide barcode sequence using the ligation product as a sequencing template.

3. The method according to claim 1 or 2, comprising a step of concentrating the target sequence in the nucleic acid sample and a step of concentrating the nucleotide barcode sequence.

Wherein said enrichment step is performed using capture or amplification, said amplification being selected from one-step PCR, two-step PCR, ligation and PCR following primer extension, circularization-based amplification, and nanopore sequencing. The method according to claim 3, wherein

To the target sequence of the sample, comprising the steps of depositing step to deposit a sample-specific pooled bar code, or a sample-specific pooled bar code, said comprising the nucleotide barcode sequence before Symbol sample 5. The method of any one of claims 1-4 , comprising the step of attaching the same sample-specific pooling barcode to nucleic acids.

A collection of carriers, which are substrates or containers, containing nucleic acids for labeling articles, comprising:
Each carrier contains at least two nucleotide barcode nucleic acids in addition to the sample nucleic acid for labeling,
Each nucleotide barcode nucleic acid comprises a different minimal nucleotide barcode sequence of at least 4 nucleotides in length,
Wherein at least two of said different nucleotide barcode nucleic acids have the same length minimal nucleotide barcode sequence,
The combination of these different nucleotide barcode nucleic acids produces a movable molecular identification barcode, whereby each such movable molecular identification barcode is different for each of the carriers in the collection,
One or both sides of the minimal nucleotide barcode sequence in the at least two nucleotide barcode nucleic acids flanked by non-viral and non-bacterial nucleotide barcode sequence identifier sequences that are identical in all nucleotide barcode nucleic acids. And/or is flanked on one or both sides by non-viral and non-bacterial extracted sequences that are identical in all nucleotide barcode nucleic acids, which enable the nucleotide barcode sequences to be distinguished,
A collection of carriers, each carrier comprising a macroscopic barcode label corresponding to the movable molecular identification barcode affixed to the carrier.

7. The collection of carriers of claim 6, wherein said collection comprises at least 100 carriers, or at least 10,000 carriers.

8. The collection of claim 6 or 7, wherein the nucleotide barcode nucleic acid for labeling does not include a sequence for transcribing the nucleic acid into RNA.

The collection of carriers according to any one of claims 6 to 8, wherein the sample is a DNA-containing sample or an RNA-containing sample .

The minimal nucleotide barcode sequence is flanked by oligonucleotide sequences for enriching the minimal nucleotide barcode sequence by amplification, or
One or more oligonucleotide-binding sequences flank one or both sides of the minimal nucleotide barcode sequence,
It allows the sequence capture based on hybridization in one or both oligonucleotide binding sequence in said nucleotide barcode sequence, collection of carriers according to any one of claims 6-9.

The collection of carriers according to any one of claims 6 to 10 , wherein the nucleotide barcode nucleic acid is contained in a fragment of a cloning vector .

The vector or vector fragment, comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, or the vector or the vector fragment, comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 11, to claim 1 1 A collection of the described carriers.

The collection of carriers according to any one of claims 6 to 12 , further comprising one or more primers for capturing and/or amplifying the nucleic acid containing the nucleotide barcode sequence.

A method of making a collection of carriers, which is a substrate or container, comprising a moveable molecular identification barcode, comprising:
a) providing a first collection of different nucleotide barcode nucleic acids comprising a minimum nucleotide barcode sequence of at least 4 nucleotides,
At least two of said different nucleotide barcode nucleic acids have minimal nucleotide barcode sequences of the same length,
The minimal nucleotide barcode sequence is between the nucleotide barcode nucleic acids in the collection, as well as one or more non-viral and non-bacterial nucleotide barcode sequence identifier sequences and/or one or more non-viral And a non-bacterial extracted sequence, which allows the identification of said minimal nucleotide barcode sequence in a nucleotide barcode nucleic acid.
b) A carrier, wherein at least two combinations of said nucleotide barcode nucleic acids of step a) are added to each carrier, each carrier having its own movable molecular identification barcode defined by the difference in the mix of the minimal nucleotide barcode sequences. Obtaining a collection of
c) labeling each carrier with a macroscopic barcode label corresponding to the movable molecular identification barcode defined by a different mix of the minimal nucleotide barcode sequences,
d) preserving the relationship between the macroscopic label and a mix of minimal nucleotide barcode sequences in the movable molecular identification barcode.
Including the method.

One or both sides of the minimal nucleotide barcode sequence in the at least two nucleotide barcode nucleic acids flanked by non-viral and non-bacterial nucleotide barcode sequence identifier sequences that are identical in all nucleotide barcode nucleic acids. and / or one or both sides, non-viral and non-bacterial extraction sequence is identical in all of the nucleotides barcode nucleic acid adjacent a method according to claim 14.

The nucleotide barcode nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, or the nucleotide barcode nucleic acid comprises a sequence of SEQ ID NO: 1 and SEQ ID NO: 11, 16 . the method of.

A method of making a collection of hosts having a vector comprising a nucleotide barcode sequence, the method comprising:
a) providing a first collection of nucleic acid vectors in a host,
The vector comprises a nucleotide barcode sequence having a minimum nucleotide barcode sequence of at least 4 nucleotides in length that differs between the nucleotide barcode nucleic acid vectors in the collection,
At least two of said different nucleotide barcode sequences have minimal nucleotide barcode sequences of the same length, and on one or both sides of said minimal nucleotide barcode sequence, one or more non-viral and non-bacterial nucleotide barcodes. A coding sequence identifier sequence, and/or one or more non-viral and non-bacterial extracted sequences, which allow the identification of said minimal nucleotide barcode sequence,
b) preparing individual colonies of the host and sequencing the nucleotide barcode sequences in the nucleic acid vector on multiple colonies to obtain a second collection of isolated colonies. There
Each colony comprises a nucleic acid vector having a different nucleotide barcode sequence,
Including the method.

18. The method of claim 17 , wherein the vector is a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, or the vector comprises the sequences of SEQ ID NO: 1 and SEQ ID NO: 11 .

A method of making a collection of carriers, which is a substrate or container, comprising a moveable molecular identification barcode, comprising:
a) providing a collection of hosts according to claim 17 ,
b) isolating the vector from the colony for colony selection,
c) adding to each carrier in said collection of carriers a combination of at least two nucleic acid vectors having different nucleotide barcode sequences,
In order to obtain a collection of carriers, each of which has at least two of said nucleic acid vectors has a unique movable molecular identification barcode, defined by the difference in the mix of the minimal nucleotide barcode sequences between the carriers, of step b) Having a minimal nucleotide barcode sequence of the same length,
Fragmenting the vector by restriction enzyme, a step of not fragment the vector by step or restriction enzymes,
d) labeling each carrier with a macroscopic barcode label corresponding to the transferable molecular identification barcode, defined by a mix of the different minimal nucleotide barcode sequences.
e) preserving the relationship between the macroscopic barcode label and the sequence of the minimal nucleotide barcode sequence in the moveable molecular identification barcode.
Including the method.

A method of tracking a nucleotide barcode nucleic acid in a set of carriers, comprising:
A step of providing a plurality of carriers, which comprises at least two nucleotide barcode nucleic acids for labeling the carrier, and is free of genomic DNA or RNA,
Each of these at least two nucleotide barcode nucleic acids comprises a minimal nucleotide barcode sequence that differs by at least 4 nucleotides in length,
The combination of these different nucleotide barcode nucleic acids produces a movable molecular identification barcode such that each movable molecular identification barcode is different for each of the carriers,
One or both sides of the minimal nucleotide barcode sequence in the at least two nucleotide barcode nucleic acids flanked by non-viral and non-bacterial nucleotide barcode sequence identifier sequences that are identical in all nucleotide barcode nucleic acids, One or both sides are flanked by non-viral and non-bacterial extracted sequences that are identical in all nucleotide barcode nucleic acids, allowing these minimal nucleotide barcode sequences to be distinguished,
Each carrier comprises a macroscopic barcode label corresponding to the moveable molecular identification barcode attached to the carrier, and
A step of sequencing a target sequence in the nucleotide barcode nucleic acid, including the minimal nucleotide barcode sequence, by a parallel sequencing method, comprising:
The parallel sequencing method is advanced by the advanced by pooling target sequence of nucleotides barcode nucleic step or, pooling the target sequence of said nucleotide barcode nucleic acid comprising a pre-Symbol Min nucleotide barcode sequence , Process,
From the obtained sequence data, a step of identifying and selecting the sequence derived from the nucleotide barcode nucleic acid, wherein the step of selecting those sequences derived from the nucleotide barcode nucleic acid from the obtained sequence data Including,
The selecting step comprises identifying a sequence having one or more nucleotide barcode sequence identifier sequences adjacent to the minimal nucleotide barcode sequence, and
Identifying and selecting said minimal nucleotide barcode sequence within said isolated sequence having a nucleotide barcode sequence identifier sequence,
Select the sequences that are between the two extracted sequences with a defined length, or select the sequences that are adjacent to one extracted sequence with the defined length, and select these selected sequences with the minimum nucleotides. A step including a step of specifying as a barcode sequence, and
Matching the identified minimal nucleotide barcode sequence with the predicted minimal nucleotide barcode sequence based on the macroscopic barcode label provided by the carrier;
Including the method.